2 beta-azido-17 beta-dialkylaminoalkylamino-5 alpha-androstan-3 alpha-ols,derivatives thereof and intermediates thereto

ABSTRACT

The above-captioned compounds are produced from the appropriate 17-keto steroid by reaction with a dialkylaminoalkylamine; the imine thus afforded is reduced to the amine, which can be formylated to yield the formamide derivative. The instant compounds are useful as pharmacological agents as is evidenced by their anti-ulcerogenic, anti-viral, and anti-microbial activity.

United States Patent Klimstra 51 Sept. 12, 1972 2 BETA-AZIDO-l7 BETA- DIALKYLAMINOALKYLAMINO-S ALPHA-ANDROSTAN-3 ALPHA-OLS, DERIVATIVES THEREOF AND INTERMEDIATES THERETO Paul D. Klimstra, Northbrook, II]. 60062 Assignee: G. D. Searle & Co., Chicago, Ill.

Filed: March 9, 1971 Appl. No.: 122,527

Inventor:

US. Cl. ..260/349, 260/3975, 424/241 Int. Cl ..C07c 117/00 Field of Search ..lMachine Searched Steroids References Cited UNITED STATES PATENTS 4/l963 Counsell et al. ..260/239.5

3,133,915 5/1964 Counsel] et al. ..260/239.5 3,238,194 3/1966 Klimstra et a1. ..260/239.5 3,326,758 6/1967 lrmscher et al 167/65 Primary Examiner-Henry A. French Attorney-John M. Brown, John J. Kolano, Elliot N. Schubert, Lowell C. Berstedt, Sybil Meloy, Walter C. Ramm and Helmuth A. Wegner [57] ABSTRACT 4 Claims, No Drawings 2 BETA-AZIDO- l 7 BETA- DlALKYLAMlNOALKYLAMINO-S ALPHA- ANDROSTAN-3 ALPHA-L8, DERIVATIVES THEREOF AND INTERMEDIATES THERETO The present invention is concerned with novel amino substituted steroids of the androstane family, and more particularly, with 2B-azidol 7B-dialkylaminoalkylamino-Sa-androstan-3a-ols and the corresponding N-formyl derivatives. These compounds can be represented by the following structural formula I N-Alk-N- (lower alkyl) 1 3 tetramethylene, pentamethylene and the branched-' chain isomers thereof.

The novel compounds of this invention can be prepared conveniently from 2B-azido-3B-hydroxy-5aandrostan-l7-one, which compound together with its method of manufacture are disclosed in US. Pat. No. 3,238,194. When this starting material istreated with a dialkylaminoalkylamine, the corresponding imine is produced. For example, the condensation of ZB-azido- 3a-hydroxy-5a-androstan-17-one with 2- diisopropylaminoethylamine in the presence of an acid catalyst such as p-toluenesulfonic acid affords 2,8- azido- 1 7-[N-( 2-diisopropylaminoethyl)iminoJ-Sa-androstan-3a-ol. Reduction of these novel imines with a metallic hydride yields the corresponding amine. This is illustrated by the reduction of the aforementioned imine with sodium borohydride in methanol, thus yielding 2B-azido-l7B-[N-(2- diisopropylaminoethyl)amino]a-androstan-3a-ol.

Production of the novel formamides of this invention is preferably accomplished by formylation of the amines with an appropriate formylating agent. One such agent is formic anhydride which can be conveniently prepared in situ from formic acid and acetic anhydride. In this manner, 2B-azido-l7B-[N-(2- diisopropylaminoethyl)amino]5a-androstan-3a-ol, when contacted with formic acid and acetic anhydride, affords 2B-azido-l7B-[N-(2- diisopropylaminoethyl)foramidoih-Sa-androstan-3a-ol.

Evidence for the anti-bacterial activity of the instant compounds is obtained from the following assay:

A mixture of 5 mg. of the test compound with 5 ml. of sterile nutrient broth is heated at C. for 20 minutes, then cooled'to about 25C. and finally serially diluted and mixed with sufficient quantities of a mixture of sterile nutrient broth and 1 percent of a culture of Bacillus subtilis to produce concentrations of approximately 400, 100, 25, and 6 mcg. of compound per ml. The resulting mixtures are incubated for 20-24 hours at 37C. Controls are provided by concurrent incubations identical with the foregoing excepting that no compound is present. Activity is determined by gross examination and potency is expressed as the minimum concentration in mcg. of compound per ml. at which no growth of the test organism is discernible.

Further evidence for the anti-bacterial activity of the instant compounds is obtained from the following assay:

Nutrient broth (manufactured by Baltimore Biological Laboratories or Difco) is prepared at twice the concentration recommended by the manufacturer, sterilized and inoculated with 2 percent (by volume) of a culture of Erwinia sp. Meanwhile the test compound is heated in sterile distilled water at a concentration of 2 mg. per ml. and a temperature of 80C. for a period of 20 minutes. An equivolume mixture of this compound preparation and the inoculated broth is incubated aerobically at 37C., then examined grossly for growth of the test organism. The incubation period is 24-48 hours. If growth of the test organism is observed, the compound is considered inactive. If no such growth is observed, the incubated mixture is serially diluted and mixed with an inoculated broth of the same composition as before excepting that the concentration is halved and 1 percent (by volume) of the culture instead of 2 percent is incorporated. Amounts of the latter broth are added such that the concentrations of 100, 10, and l mcg. of compound per ml. result. The mixtures thus obtained are incubated as before, then examined grossly for growth of the test organism. Potency is expressed as the minimum concentration at which no growth of the test organism is discernible. Controls are provided by concurrent incubations identical with the foregoing except for absence of the test compound.

The compounds of this invention display anti-fungal activity as determined by the following test procedure:

The test compound is dissolved or suspended in melted Sabouraud agar and is held at 80 C. for 20 minutes. Dilutions are made from this preparation in melted Sabouraud agar in order to yield concentrations of the test substance of 1000, 100, 10 and l mcg./ml. in the agar. The agar is permitted to cool and solidify and is then surface inoculated with a suspension of spores of Verticillium albo-atrum. The inoculated media are incubated at room temperature for 6-7 days, then are examined grossly for the presence or absence of growth of the test organism. Control preparations lacking the test compound are employed for comparative purposes. The activity of the compound is reported as mcg.

of the compound/ml. of agar which completelyprevents visible growth of the test organism.

Evidence of the anti-protozoa] activity of the instant compounds is provided by a standardized test for their tion of 2,000 7 per ml. and a temperature of 80 C. for 20 min. An equivolume mixture of this compound preparation and the inoculated medium is incubated aerobically at 32 C. for 48 hr. and then examined microscopically for the presence of motile tetrahymena. If any are observed, the compound is considered inactive. If no motile tetrahymena are observed, the incubated mixture is serially diluted and mixed with an inoculated medium of the same composition as that described above excepting that 1,000 parts of distilled water instead of 500 parts and percent (by volume) of the culture instead of 10 percent are incorporated. Amounts of the latter medium added are such that concentrations of 100, 10, and l 'y of compound per ml. result. The mixtures thus obtained are incubated as before and then examined microscopically for motile tetrahymena. Potency is expressed as the a minimum concentration at which no motile tetrahymena are discernible. Controls are provided by concurrent incubations identical with the foregoing except for the absence of compound.

The present compounds are further useful as antiviral agents as demonstrated by the fact that they inhibit the growth of influenza virus type A (strain 575). This is demonstrated by the following test procedure. Cell cultures of primary Rhesus monkey kidney maintained in 25 cc. plastic flasks and each containing test compound at concentrations of 625, 125, 25, 5 or 1 microgram per milliliter are prepared in pairs. These flasks, and an identical pair of flasks containing no test compound, are each inoculated with a dose of influenza virus type A (strain 575) previously shown to produce maximum hemadsorption and minimum cytopathogenic effects after a 24 hour incubation. Where the cultures contain test compound, the virus is added one hour after addition of the compound to the culture; After 24 hours incubation of the cultures, the supernatant fluids are removed and 3.0 ml. of a 0.4 percent suspension of guinea pig erythrocytes are added to each flask. The flasks are then incubated at 4C. in a horizontal position for 30 minutes. The flasks are rocked every 10 minutes during the incubation period. After this incubation, the red cell suspension is decanted from each flask, the flasks are washed twice with 3.0 ml of phosphate buffer solution (pH 7.4) to remove unadsorbed red cells, and 3.0 ml. of distilled water is then added to lyse the adsorbed cells. The flasks are then further incubated at 37C. for 30 minutes in a horizontal position and the flasks are rocked every 10 minutes. After this incubation, the fluid contents of the pairs of flasks are combined to form an assay unit and are placed at room temperature for -30 minutes to allow settling of cellular debris. A pair of control flasks identical with the above, except for the absence of test compound and virus inoculation, are run concurrently. The resulting hemoglobin solutions from each assay unit are then read for optical density in a Beckman spectrophotometer at about 415 millimicrons. A test compound is considered active if, at

one of the tested levels, it reduces the optical density reading by at least 50 percent relative to the virus control.

The invention will appear more fully from the examples which follow. These examples should not be construed as limiting the invention either in spirit or in scope as numerous modifications will be apparent to those skilled in the art. Quantities of material are presented in parts by weight unless otherwise noted and temperatures are given in degrees Centigrade (C.

EXAMPLE 1 GH(CHs)2 N-CHrCHr-N EXAMPLE 2 To a solution of 25 parts of 2,6-azido-l7-[N-(2- diisopropylaminoethyl)imino]5a-androstan-3a-ol in 277 parts of methanol is added 13 parts of sodium borohydride with stirring over a 15 minute period. The solution is stirred for an additional one-half hour and then water is added and the solution cooled. The resulting crude produce which precipitates is collected by filtration, washed with water and partially air dried. Then the crude product is dissolved in hot acetone, and the hot acetone solution is filtered through diatomaceous earth. The pure product, 2B-azido-17B-[N-( 2- diisopropylaminoethyl)amino]5a-androstan-3a-ol is obtained upon recrystallization from acetone and displays on optical rotation, in chloroform, of +30.05.

This compound is represented by the following structural formula H CH(CHa)z NCHGCHZ CH3 CH(CH3)2 EXAMPLE 3 Substitution of an equivalent quantity of 3- dimethylaminopropylamine in the procedure of Example 1 yields ZB-azidol 7B-[N-( 3- dimethylaminopropyl)imino]-5a-androstan-3a-ol.

EXAMPLE 4 EXAMPLE 5 8.0 Parts of 2,8-azido-l7B-[N-( 2- diisopropylaminoethyl)amino]5a-androstan-3a-ol is treated with a mixture comprised of 73.2 parts of formic acid and 64.9 parts of acetic anhydride. After the reaction mixture is heated on a steam bath for 2 hours and then allowed to remain at room temperature for l l hours, it is neutralized by the addition of 25 percent aqueous sodium hydroxide. During the neutralization the mixture is cooled and methanol is added to retain homogeneity. Water is then added with cooling and a granular material precipitates. The precipitate is collected by filtration, washed with water, and air dried. Recrystallization of the precipitate from ethyl acetate affords pure ZB-azido-l 7B-[N-( 2- diisopropylaminoethyl)formamido]-5a-androstan-3aol, which exhibits an optical rotation, in chloroform, of +10.34 and is represented by the following structural formula EXAMPLE 6 By substituting an equivalent quantity of ZB-azidol 7 ,B-[N-(S-dimethylaminopropyl)amino]-5a-androstan- 311-01 and otherwise following the procedure of Example 5, 2,8-azido- 1 7B-[ N-( 3-dimethylaminopropyl)formamido]-5a-androstan-3a-ol is produced.

What is claimed is:

l. A compound of the formula wherein R is selected from the group consisting of hydrogen and a formyl radical and Alk is a lower alkylene radical.

2. As in claim 1, the compound which is ZB-azido-l 7 /3-[N-(2-diisopropylaminoethyl)aminol-5a-androstan- 3. As in claim 1, the compound which is 2,3-azido-l7 ,B-[N-( 2-diisopropylaminoethyl)formamido1-5a-androstan-3a-ol.

4. 2,8-azidol 7-[ N-( 2-diisopropylaminoethyl)imino] 

2. As in claim 1, the compound which is 2 Beta -azido-17 Beta -(N-(2-diisopropylaminoethyl)amino)-5 Alpha -androstan-3 Alpha -ol.
 3. As in claim 1, the compound which is 2 Beta -azido-17 Beta -(N-(2-diisopropylaminoethyl)formamido)-5 Alpha -androstan-3 Alpha -ol.
 4. 2 Beta -azido-17-(N-(2-diisopropylaminoethyl)imino) -5 Alpha -androstan-3 Alpha -ol. 